1. Field of the Invention
The invention relates to methods for diagnosing B-cell lymphoma in an animal. In particular, the invention relates to methods for distinguishing an animal having diffuse large B-cell lymphoma (DLBCL) with an activated B-cell (ABC) phenotype from an animal having DLBCL with a non-activated germinal-center (GC) phenotype. The invention also relates to methods for identifying compounds for treating B-cell lymphoma. The invention further relates to reagents and methods for determining the amount of miR-155 in sample isolated from an animal. In this regard, the invention relates to a set of oligonucleotides for determining the amount of miR-155 in sample isolated from an animal.
2. Background of the Invention
Inappropriate expression of proto-oncogenes or inactivation of tumor supressor genes can contribute to cancer. One example is the BIC gene, which was originally identified as a common site for insertion of pro-viral DNA in avian leukosis virus (ALV)-induced lymphomas (Tam et al., 1997, Mol. Cell. Biol. 17:1490-502; Clurman et al., 1989, Mol. Cell. Biol. 9:2657-64). Activation of the BIC gene can accelerate the pathogenesis of lymphomas and leukemias that are associated with up-regulation of c-MYC, showing that BIC functions in the etiology of these diseases (Tam et al., 2002, J. Virol. 76:4275-86). Expression of BIC RNA is low in normal lymphoid tissues, but elevated in Hodgkin and children's Burkitt lymphoma and in in vitro activated B- and T-cells (Haasch et al., 2002, Cell. Immunol. 217:78-86; Metzler et al., 2004, Genes Chromosomes Cancer 39:167-69; van den Berg et al., Genes Chromosomes Cancer 37:20-28).
Avian, murine, and human BIC RNA is a spliced and polyadenylated transcript that is approximately 1.7 kb in length (including the poly A tail) and is presumably generated by RNA polymerase II. Because BIC transcripts lack long open reading frames (ORFs), and their short, putative ORFs are not conserved, it has been suggested that BIC RNA functions as a non-protein-coding RNA (Tam, 2001, Gene 274:157-67). Recently, a mouse microRNA (miRNA) molecule, designated miR-155 (Lagos-Quintana et al., 2002, Curr. Biol. 12:735-39), was found to be encoded within the only phylogenetically conserved region of BIC RNA (Id.). Typically, miRNAs are ˜22 nucleotide long molecules that function in post-transcriptional down-regulation of gene expression in plants, vertebrates, and invertebrates (Bartel, 2004, Cell 116:281-97; He et al., 2004, Nat. Rev. Genet. 5:522-31; Pasquinelli, 2002, Trends Genet. 18:171-73). Thus, miR-155 could be responsible for the oncogenic activity attributed to BIC RNA, inter alia, by down-regulating tumor suppressor gene transcription.
In animal cells, endogenous miRNAs are produced from primary RNA polymerase II transcripts (i.e., pri-miRNAs) by sequential processing in the nucleus and cytoplasm (Cullen, 2004, Mol. Cell. 16:861-65). Nuclear precursor RNAs are cleaved by the endonuclease Drosha in a “microprocessor complex,” releasing pre-miRNAs, which are short 60-70 nucleotide imperfect hairpin structures. After transport to the cytoplasm by exportin-5, pre-miRNAs are processed by the endonuclease DICER, generating ˜22 nucleotide duplexes, one strand of which is the mature miRNA. The conserved region of BIC RNA encoding miR-155 can form an imperfect hairpin structure (Tam, 2001, supra), suggesting that miR-155 is generated by this pathway (FIG. 1).
Changes in the levels of miRNAs may alter control of growth or apoptosis in some cancers (McManus, 2003, Semin. Cancer Biol. 13:253-58; Xu et al., 2004, Trends Genet. 20:617-24). Reductions in the levels of miR-15a plus miR-16, let-7a, and miR-143 plus miR-145 have been reported in chronic lymphocytic leukemia (CLL) (Calin et al., 2002, Proc. Natl. Acad. Sci. U.S.A. 99:15524-29), lung cancer (Takamizawa et al., 2004, Cancer Res. 64:3753-56), and colon carcinoma (Michael et al., 2003, Mol. Cancer Res. 1:882-91), respectively. Although BIC RNA is up-regulated in some human lymphomas (Metzler et al., 2004, supra; van den Berg et al., 2003, supra), very little is known about the levels of miR-155 in these cancers.
Diffuse large B-cell lymphoma (DLBCL), an aggressive B-cell neoplasm accounting for 30-40% of all lymphoma cases (The Non-Hodgkin's Lymphoma Classification Project, 1997, Blood 89:3909-18), can be categorized immunohistochemically into groups with significantly different clinical outcomes (Chang et al., 2004, Am. J. Surg. Pathol. 28:464-70). The prognosis is poorer for patients having DLBCL with an activated B-cell (ABC) phenotype than a non-activated germinal-center (GC) phenotype. So far, a relationship has not been examined between miR-155 and BIC RNA levels and the phenotypes of this most frequent of all lymphoid neoplasms.
Thus, there is a need in the art for methods for diagnosing B-cell lymphoma in patients. In addition, there is a need in the art for methods for distinguishing individuals having DLBCL with an ABC phenotype from individuals having DLBCL with a GC phenotype. Such methods would be particularly useful in situations where conventional histologic and immunophenotypic methods cannot provide an accurate diagnosis of B-cell lymphoma, and in particular, DLBCL with an ABC phenotype. Such methods would also be useful in determining suitable courses of therapy for treating patients having B-cell lymphoma, and in particular, DLBCL with an ABC phenotype. Therefore, the development of such diagnostic methods would have wide application in the medical arts.